The cryo-EM structure of homotetrameric attachment glycoprotein from langya henipavirus

Langya Henipavirus (LayV) infection is an emerging zoonotic disease that has been causing respiratory symptoms in China since 2019. For virus entry, LayV’s genome encodes the fusion protein F and the attachment glycoprotein G. However, the structural and functional information regarding LayV-G remains unclear. In this study, we revealed that LayV-G cannot bind to the receptors found in other HNVs, such as ephrin B2/B3, and it shows different antigenicity from HeV-G and NiV-G. Furthermore, we determined the near full-length structure of LayV-G, which displays a distinct mushroom-shaped configuration, distinguishing it from other attachment glycoproteins of HNV. The stalk and transmembrane regions resemble the stem and root of mushroom and four downward-tilted head domains as mushroom cap potentially interact with the F protein and influence membrane fusion process. Our findings enhance the understanding of emerging HNVs that cause human diseases through zoonotic transmission and provide implication for LayV related vaccine development.

For the biochemical assay, each experiment was replicated at least 3 times on separate occasions.Experimental findings were reliably reproduced.For experiments of the LayV-G and NiV-G binding to ephrinB2 and ephrinB3 were pertormed four times, all attempts showed similar results.Multiple cryo-EM grids were prepared and frozen, datas were collected for two or three selected grids of the full-length LayV-G structures.Representative ones of protein purification and micrographs were shown in the supplementary information.Flow cytometry experiments were performed independently in duplicate.All ELISA assays were performed three times independently.
Randomization is not relevant for this study, as there no groups allocated in any of the experiments.This study did not involve animals or humans research participants.
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Flow Cytometry
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A numerical value for number of cells or percentage (with statistics) is provided.Full length Ephrin B2 (EAX09085.1)or Ephrin B3 (NP_001397.1) was stably transfected into CHO cells using the lentivector with GFP as a marker to monitor gene expression.Briefly, pCDH-Ephrin B2 or B3 and envelope vectors were co-transfected into packaging cells.The cell culture supernatants containing the lentivirus were harvested 48 hours later and further incubated with CHO cells.Afterward, stable CHO-EphrinB2 or B3 cell lines were produced via puromycin selection.

Methodology
The CHO-Ephrin B2 or CHO-Ephrin B3 cells were incubated with various HNV G protein (20 !g/mL) at 4°C for 30 min.Afterwards, 50 !L of diluted SureLight® APC Anti-6X His tag (Abcam, Cat# ab72579) was added to the cells and incubated at 4°C for 30 min.Finally, cells were washed with PBS and resuspended after centrifuged.

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The gating strategy is firstly gating the major population according to FSC-A and SSC-A, then exclude of doublets and dead cells(BV421 positive population), finally analyzing the mean fluorescence intensity of APC in GFP-positive population.
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